Oral squamous carcinomas (OSCC) are characterized by complex, often near-triploid karyotypes with multiple numerical and structural abnormalities. The frequency of aneuploidy in oral cancers is reportedly higher than uterine, cervical, colorectal or breast cancers. In most OSCC cell lines, cells display structural and numerical variations on a background of clonal chromosomal alterations. Thus, chromosomal instability (CIN) may play a key role in OSCC, making it an excellent test system. What is the cause of this high rate of CIN and resulting aneuploidy in oral cancer? We hypothesize that alterations in key spindle, centrosomal, and centromeric (cytoskeletal) proteins are an important contributing factor to the CIN in OSCC. Our preliminary results support our hypothesis. Examination of OSCC cells reveals multipolar spindles, lagging chromosomes, abnormal spindle structure, and a high frequency of micronuclei. To investigate the mechanism of CIN in OSCC, we will compare our OSCC cell lines to normal human oral keratinocyte (NHOK) cultures to satisfy the following Specific Aims, to 1) sequentially characterize the cytogenetic and cytoskeletal alterations in the same OSCC cells compared to control NHOKs by immunocytochemistry with antibodies to key cytoskeletal proteins and classical molecular cytogenetic analyses, 2) define a basis for the cytoskeletal defects in OSCC in terms of altered expression of selected cytoskeletal proteins and/or aberrations in their genes using blotting and sequencing, and (3) determine whether the observed changes in cytoskeletal proteins are causally related to CIN by altering the levels of the cytoskeletal proteins in NHOKs and assessing whether this change causes CIN. This study is expected to produce the first clear-cut evidence that cytoskeletal dysfunction is a major contributor to the observed CIN in OSCC, will clarify the manner in which segregation is altered in OSCC cells, and will identify specific cytoskeletal proteins, altered expression of which may contribute to the CIN in OSCC. Altered expression of one or more of these proteins may also serve as a useful biomarker of CIN in oral mucosal or tumor cells, possibly for early detection. If re-establishing normal mitotic protein levels corrects segregation defects, it may prove to be an effective means of minimizing CIN in tumors, thus, heterogeneity with numerous, varied, irreparable genetic alterations, may be fruitless. Identification of cells bearing CIN will then enable application of therapies that guide the cells into a natural cell death.